1. Field of the Invention
This invention is directed generally to assays involving blood clotting and clot removal and more particularly to assays for fibrinolytic activity.
2. Description of the Background
Fibrinolysis is the mechanism by which thrombin-generated fibrin clots are removed. The major components of the fibrinolytic system are plasminogen, plasminogen activators and inhibitors, and plasmin inhibitors. Plasminogen is the proenzyme in plasma, which upon conversion to its active form, plasmin, is considered primarily responsible for the digestion of fibrin clots.
Methods in this area may be broadly divided into bioassays for plasmin activity and immunoassays for the presence of various antigenic forms of tissue plasminogen activator (t-PA) or other component antigens of the fibrinolysis system. The purification and subsequent cloning of t-PA has allowed standardization of the inoculum for antibody preparations, but the diverse epitopes against which monoclonal antibodies have been raised make standardization of immunoassays using these reagents very difficult. The bioassay approach in which plasmin is measured is also subject to variation in that a number of different substrates are used, influencing the plasmin potency estimates.
For example, one bioassay method based on the lysis of fibrin creates a fibrin gel in a Petri dish through the addition of thrombin to fibrinogen. Then either purified fibrinolytic system components or plasma is added to the surface of the matrix, and the diameter of the zone of lysis is measured after 18 to 20 hours. This method is relatively slow, is not particularly accurate, and does not distinguish between intrinsic and extrinsic activators.
The bioassay method most often described in the literature is the chromogenic assay technique. In this technique a p-nitroaniline-linked tripeptide specific for the protease of interest is hydrolyzed, releasing the p-nitroaniline and resulting in a color change which can be followed spectrophotometrically. When this technique is applied to t-PA detection, however, a fibrin substitute is also needed to induce t-PA binding and consequent activity enhancement.
Immunological assays for components of the fibrinolytic system are generally applications of the normal ELISA or radiometric immunoassays. These systems fail to give an accurate measure of the activity of the system in vivo. Recently, bioimmunoassays have been developed which combine the immobilization features of the immunoassay approach with the functional aspects of the chromogenic assays. These bioassays do not utilize fibrin as the substrate or as a t-PA activity enhancer.
Recombinant t-PA offers a promising approach to coronary thrombolysis in patients with acute myocardial infarction due to coronary thrombosis. With the use of t-PA to achieve specific thrombolysis, assay systems for all aspects of the fibrinolytic system which are standardized, simplified, and readily available are needed. There is, therefore, a significant need for providing rapid and efficient assays which allow for high sensitivity, rapidity, and which are capable of automation for detecting a wide variety of factors of interest.